Depletion of embryonic stem cell signature by histone deacetylase inhibitor in NCCIT cells: involvement of Nanog suppression.

Abstract

The embryonic stem cell-like gene expression signature has been shown to be associated with poorly differentiated aggressive human tumors and has attracted great attention as a potential target for future cancer therapies. Here, we investigate the potential of the embryonic stem cell signature as molecular target for the therapy and the strategy to suppress the embryonic stem cell signature. The core stemness gene Nanog is abnormally overexpressed in human embryonic carcinoma NCCIT cells showing gene expression profiles similar to embryonic stem cells. Down-regulation of the gene by either small interfering RNAs targeting Nanog or histone deacetylase inhibitor apicidin causes reversion of expression pattern of embryonic stem cell signature including Oct4, Sox2, and their target genes, leading to cell cycle arrest, inhibition of colony formation in soft agar, and induction of differentiation into all three germ layers. These effects are antagonized by reintroduction of Nanog. Interestingly, embryonic carcinoma cells (NCCIT, NTERA2, and P19) exhibit a higher sensitivity to apicidin in down-regulation of Nanog compared with embryonic stem cells. Furthermore, the down-regulation of Nanog expression by apicidin is mediated by a coordinated change in recruitment of epigenetic modulators and transcription factors to the promoter region. These findings indicate that overexpression of stemness gene Nanog in NCCIT cells is associated with maintaining stem cell-like phenotype and suggest that targeting Nanog might be an approach for improved therapy of poorly differentiated tumors.

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Depletion of Embryonic Stem Cell Signature by Histone Deacetylase
Inhibitor in NCCIT Cells: Involvement of Nanog Suppression
Jueng Soo You,
Yae Jee Jeon,
1Jae Ku Kang,
1Eun Jung Cho,
2Dong-Wan Seo,
1and Jeung-Whan Han
3Jae Hyun Park,
1
1Jong Woo Park,
1Jae Cheol Lee,
1
1Department of Biochemistry and Molecular Biology, College of Pharmacy, Sungkyunkwan University, Suwon, Korea;
Pharmacology, College of Medicine, Konyang University, Daejeon, Korea; and
Kangwon National University, Chuncheon, Korea
2Department of
3Department of Molecular Bioscience,
Abstract
The embryonic stem cell-like gene expression signature has
been shown to be associated with poorly differentiated
aggressive human tumors and has attracted great attention
as a potential target for future cancer therapies. Here, we
investigate the potential of the embryonic stem cell signature
as molecular target for the therapy and the strategy to
suppress the embryonic stem cell signature. The core stemness
gene Nanog is abnormally overexpressed in human embryonic
carcinoma NCCIT cells showing gene expression profiles
similar to embryonic stem cells. Down-regulation of the gene
by either small interfering RNAs targeting Nanog or histone
deacetylase inhibitor apicidin causes reversion of expression
pattern of embryonic stem cell signature including Oct4, Sox2,
and their target genes, leading to cell cycle arrest, inhibition of
colony formation in soft agar, and induction of differentiation
into all three germ layers. These effects are antagonized by
reintroduction of Nanog. Interestingly, embryonic carcinoma
cells (NCCIT, NTERA2, and P19) exhibit a higher sensitivity to
apicidin in down-regulation of Nanog compared with embry-
onic stem cells. Furthermore, the down-regulation of Nanog
expression by apicidin is mediated by a coordinated change in
recruitment of epigenetic modulators and transcription
factors to the promoter region. These findings indicate that
overexpression of stemness gene Nanog in NCCIT cells is
associated with maintaining stem cell-like phenotype and
suggest that targeting Nanog might be an approach for
improved therapy of poorly differentiated tumors. [Cancer
Res 2009;69(14):5716–25]
Introduction
Phenotypic and functional similarities between tumor cells and
normal stem cells have generated great interest in the possible
links between these classes of cells (1). Both tumor cells and
normal stem cells have extensive proliferative potential and
the ability to give rise to new tissues, although normal stem cells
do so in a highly regulated manner, whereas tumor cells self-renew
in a poorly regulated manner and differentiate abnormally.
Based on these similarities, it is hypothesized that tumors might
arise from normal stem or progenitor cells (2). However, it is
still possible that tumor cells can acquire stem cell-like character-
istics through progressive dedifferentiation during their develop-
ment. In addition, recent studies indicate that only a small
subpopulation of tumor cells are clonogenic and have the exclusive
ability to regenerate a tumor mass (3). These clonogenic cells have
been found in a wide array of highly undifferentiated tumors
including blood, brain, breast, and colon cancers with the
capability of both self-renewal and at least partial differentiation,
similar to that of normal stem cells (4). These characteristics of the
cells have led to their designation as cancer stem cells or tumor-
initiating cells with stem cell-like properties. Although some
regulators of the stem cell function have been shown to be
implicated in tumorigenesis, a detailed characterization of the
activity of stem cell-associated regulatory networks in tumors is
still lacking.
The core stemness transcription factors, Oct4, Sox2, and Nanog,
which are expressed in embryonic stem cells and embryonic
carcinoma cells, are thought to be central to the transcriptional
regulatory network that specifies the identity of both cell types
(5, 6). Oct4, a member of POU class of homeodomain proteins, is
normally found in the inner cell mass of the blastocyst and is
required to maintain the pluripotency of the inner cell mass cells
(7). During differentiation, its expression is reduced through
mechanisms involving epigenetic modification. Mice lacking Oct4
exhibit early embryonic lethality due to a failure to form the inner
cell mass, indicating the critical role of Oct4 in controlling self-
renewal of embryonic stem cells. In contrast, overexpression of
Oct4 is directly linked to the tumorigenic potential of embryonic
stem cells (8). Nanog, another transcription factor, also plays a
critical role in regulating cell fate of the pluripotent inner cell mass
during embryonic development (9). In the absence of Nanog,
mouse embryonic stem cells differentiate into visceral/parietal
endoderm, whereas overexpression of Nanog can override the
requirement of leukemia inhibitory factor for self-renewal of
embryonic stem cells (10). Similarly, Nanog overexpression in
human embryonic stem cells enables maintenance of these cells in
the pluripotent state for a prolonged period, and knockdown of
Nanog promotes differentiation, indicating a role of Nanog in self-
renewal of human embryonic stem cells (11). Additionally, forced
expression of Nanog in hematopoietic stem cells followed by a
transplantation of these cells leads to development of a
lymphoproliferative disorder as well as gy T-cell malignancy (12).
Interestingly, Nanog and Oct4 are overexpressed in oral cancer
stem-like cells enriched through sphere formation by cultivating
oral squamous cell carcinoma cells (OSCC) from established OSCC
cell lines or primary culture of OSCC patients (13). Moreover, OSCC
patients positive to the expression of Nanog, Oct4, and CD133 have
been associated with the worst survival prognosis.
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Jeung-Whan Han, Department of Biochemistry and
Molecular Biology, College of Pharmacy, Sungkyunkwan University, 300 Cheoncheon-
dong, Jangan-gu, Suwon 440-746, Korea. Phone: 82-31-290-7716; Fax: 82-31-290-7796;
E-mail: jhhan551@skku.edu.
I2009 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-08-4953
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Recently, analysis of the expression patterns of gene sets
associated with the embryonic stem cell identity in various human
tumor types has identified a potential link between genes
associated with embryonic stem cell identity and the histopatho-
logic traits of tumors (14). Stemness genes such as Nanog, Oct4,
Sox2, and c-Myc are normally enriched in embryonic stem cells,
and activation targets of these genes have been shown to be
overexpressed in histologically poorly differentiated tumors,
whereas these tumors repress preferentially polycomb-related
genes. This embryonic stem cell signature has been found in
several poorly differentiated cancers and often associated with a
poor clinical outcome. These observations suggest that stem cell-
like characteristics shown by many poorly differentiated tumors
might be attributed to abnormal activation of these genes.
Small molecules could offer several advantages that include the
ability for a temporal, tunable, and modular control of specific
proteins involved in various cellular processes without abnormal
genetic modification (15). Cell-based phenotypic assay and pathway
screening of synthetic small molecules and natural products have
historically provided useful chemical tools to modulate and/or
study complex cellular processes; thereby, some small molecules
such as dexamethasone and all-trans retinoic acid have been
identified as agents inducing stem cell differentiation. Moreover,
histone deacetylase inhibitors (HDACI) and DNA demethylating
agents, which target abnormal epigenetic modifications, induce
differentiation of various transformed cells. However, the effects of
these small molecules are not fully evaluated in embryonic stem
cells or cancer stem cells. Therefore, the investigation of the
potential of embryonic stem cell signature as molecular target for
treatment of aggressive human tumors will provide a valuable
rationale to develop an effective strategy for future cancer therapy.
In this study, we report the first evidence that HDACI apicidin is
capable to down-regulate Nanog expression in poorly differentiated
human embryonic carcinoma cells, which is mediated by a
coordinated change in recruitment of epigenetic modulators
(DNMT3B, CBP, and EZH2) and transcription factors (SP1, GCNF,
Oct4, Nanog, and Sox2) to its promoter region. This down-
regulation of Nanog expression leads to decrease of other stemness
gene expression such as Oct4, Sox2, and their target genes and the
induction of differentiation markers for all three germ layers
including endoderm, mesoderm, and ectoderm. Furthermore,
sensitivity to apicidin in suppression of Nanog expression is higher
in embryonic carcinoma cells than in embryonic stem cells. This
suggests that targeting the stemness gene Nanog may provide a
novel strategy for therapy of poorly differentiated tumors
associated with the embryonic stem cell signature.
Materials and Methods
Cell culture. NCCIT cells (American Type Culture Collection) were
cultured in RPMI 1640 (Life Technologies), supplemented with 10% fetal
bovine serum (Hyclone Laboratories) and 1% penicillin/streptomycin (Life
Technologies).
Reagents and antibodies. Apicidin [cyclo(N-O-methyl-L-tryptophanyl-L-
isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)] was prepared from
Fusarium sp. strain KCTC 16677 according to the method described (16).
Commercial primary antibodies’ information is described in Supplementary
Information.
RNA extraction and reverse transcription-PCR. Total RNA was
extracted using easy-Blue reagent (iNtRON Biotechnology). PCRs were run
by using the gene-specific primers (Table S1). Detailed method and gene
specific primers are described in Supplementary Information.
Western blot analysis. Cell lysates were boiled in Laemmli sample
buffer for 3 min, and 30 Ag of each protein were subjected to SDS-PAGE.
Detailed method is described in Supplementary Information.
Gene expression profiling. Illumina’s HumanRef-8 v2 BeadChips were
used to generate expression profiles. All expression data are available at
National Center for Biotechnology Information Gene Expression Omnibus
under series no. GSE13177. The individual expression arrays are listed as
GSM333027 to GSM333030. Detailed gene expression profiling method is
described in Supplementary Information.
DNA methylation bisufite sequencing analysis. DNA was purified by
using the G-DEX genomic DNA miniprep kit (iNtRON Biotechnology).
Purified genomic DNA (1 Ag) was treated with sodium bisulfite solution.
Detailed method is described in Supplementary Information.
Chromatin immunoprecipitation assay. Chromatin immunoprecipi-
tation assays were done using the Acetyl Histone H3 Immunoprecipitation
Assay Kit (Upstate Biotechnology) according to the manufacturer’s
instructions. Detailed chromatin immunoprecipitation method is described
in Supplementary Information.
Cell proliferation, cell cycle, and apoptosis analysis assays. Cell
proliferation assay was carried out with cell counting and Bromodeoxyur-
idine Colorimetric Cell Proliferation Kit (Roche Molecular Biochemicals).
Cell cycle assays were done using Cycletest Plus DNA Reagent Kit (BD
Biosciences) according to the manufacturer’s instructions. The detail
analyzing method is described in Supplementary Information.
Soft-agar colony-forming assay. NCCITcells (1.0 ? 105) were seeded in
0.4% agar and incubated at 37jC for 21 days. Colonies were visualized by
0.005% crystal violet staining and counted.
Small interfering RNA transfection. Cells were transfected with
scrambled or target gene-specific small interfering RNA (siRNA) using
Lipofectamine 2000 (Invitrogen). siRNAs specifically targeting Nanog or SP1
were purchased from Dharmacon and/or Invitrogen.
Results
Differential expression of stemness genes in various human
cell lines. Poorly differentiated aggressive human tumors have
been shown to possess embryonic stem cell signature, which is
associated with poor prognosis (14). To examine the involvement of
the key regulators of embryonic stem cell signature in tumorigen-
esis, we have analyzed the expression level of the core stemness
genes in various human cell lines including embryonic stem H9,
embryonic carcinoma NCCIT, normal fibroblast BJ, cervical carcinoma
HeLa, breast cancer MCF7, ductal carcinoma HCC1954, osteocarci-
noma U2OS, and lung carcinoma H1299 cells. Whereas Sox2 ex-
pression is detected in all cell lines tested with different levels, Oct4
and Nanog are expressed only in both embryonic stem H9 and
embryonic carcinoma NCCIT cells (Fig. 1A). However, the level of
Nanog expression is much higher in NCCIT than H9 cells, although
Oct4isexpressedwithacomparablelevelinbothcells,indicatingthe
dysregulation of Nanog expression in NCCITcells.
Selective down-regulation of Nanog expression by HDACI
apicidin in embryonic carcinoma cells. To identify the
molecules that can suppress the abnormal Nanog expression in
NCCIT cells, these cells have been treated with small molecules,
which have been shown previously to regulate cell proliferation or
differentiation, such as all-trans retinoic acid, reversine, sonic
hedgehog signaling inhibitor (cyclopamine), mammalian target of
rapamycin inhibitor (rapamycin), clinical anticancer agents (cis-
platin, 5-fluorouracil), demethylating agent (5-aza-cytidine), and
HDACIs (apicidin, trichostatin A, and suberoylanilide hydroxamic
acid; ref. 17). Among the molecules tested, HDACIs (trichostatin A,
apicidin, and suberoylanilide hydroxamic acid) have the most
significant effect on down-regulation of Nanog mRNA and protein
in a dose-dependent manner (Supplementary Fig. S1).
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Among three HDACIs, we have further analyzed the effect of
apicidin on Nanog expression in embryonic stem and embryonic
carcinoma cells. Interestingly, apicidin treatment does not affect
Nanog mRNA expression in normal human embryonic stem cells,
whereas it causes a marked decrease in Nanog mRNA expression in
malignant human embryonic carcinoma cells (Fig. 1B). The effect
on Nanog protein expression is similar to that on Nanog mRNA
expression, except a slight increase in sensitivity of human
embryonic stem H9 cells to apicidin (Fig. 1B). Consistently,
expression levels of Nanog mRNA and protein show no alteration
in mouse embryonic stem J1 cells when treated with apicidin,
indicating that embryonic carcinoma cells are more sensitive to
apicidin treatment in inhibition of Nanog expression than
embryonic stem cells (Fig. 1C). Interestingly, down-regulation of
Nanog expression is accompanied by a decrease in Oct4 and Sox2
expression in both mouse and human embryonic carcinoma cells
(Fig. 1). In addition, apicidin treatment leads to down-regulation of
other stemness genes, such as FOXD3 and REX1 in NCCIT cells
(data not shown), and induces a time- and dose-dependent
decrease in Oct4, Nanog, and Sox2 expression in NCCIT cells
(Supplementary Fig. S2). Among the stemness genes tested, Nanog
expression is most sensitive to apicidin treatment, suggesting that
Figure 1. Differential expression of
stemness genes in various human cell
lines and apicidin-mediated down-
regulation of stemness genes in embryonic
carcinoma but not in embryonic stem cells.
A, expression levels of stemness genes
were determined by Western blot analysis
in various human cell lines. B, human
embryonic stem H9 and embryonic
carcinoma NCCIT cells were exposed
to 1 Amol/L apicidin. C, mouse embryonic
stem J1 and embryonic carcinoma P19
cells were exposed to 0.5 Amol/L apicidin
for 24 h. Cells were subjected to qPCR and
Western blot analysis (B and C) for the
expression of Nanog, Oct4, and Sox2.
D, effects of apicidin on Nanog expression
in other cancer cell lines were examined by
RT-PCR and Western blot. Representative
of three independent experiments. con,
control; api, apicidin.
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down-regulation of Oct4 and Sox2 expression might be mediated
through down-regulation of Nanog. Consistent with these results, a
selective knockdown of Nanog expression by two independent
siRNA pools corresponding various regions of the Nanog transcript
leads to a significant decrease in the expression levels of mRNAs as
well as proteins of Oct4 and Sox2, whereas the control siRNA does
not affect expression of these genes (Nanog, Oct4, and Sox2) and an
irrelevant control gene (Supplementary Fig. S3). To further confirm
the effects, we have analyzed changes of Nanog expression in two
other poorly differentiated human cancer cell lines including
NTERA2 (embryonic carcinoma line) and PC3 (a prostate
adenocarcinoma cell line). Similarly, apicidin markedly decreases
both Nanog mRNA and protein levels in NTERA2 cells, which
express Nanog at high levels (Fig. 1D). However, the Nanog mRNA
and protein in PC3 are not detected (Fig. 1D). These results
indicate that apicidin down-regulates Nanog expression in Nanog-
positive cancer cells.
Depletion of embryonic stem cell-like signature in NCCIT
cells by apicidin. To evaluate the full potential of HDACI apicidin
on transcriptional alterations in NCCITcells, global gene expression
profiles were determined by an Illumina HumanRef-8 v2 Expression
BeadChips and compared between control and apicidin-treated
NCCIT cells. Two thousand twenty-five genes of a total of 23,920
arrayed genes are affected by apicidin treatment with >2-fold signi-
ficant changes relative to control NCCITcells (P < 0.05; dataset S1).
Among these genes, 1,030 genes are classified into the 13 gene sets
associated with the human embryonic stem cell identity, which are
classified previously by Ben-Porath and colleagues (ref. 14; dataset
S2). The various embryonic stem cell-expressed and Nanog, Oct4,
and Sox2 target gene sets are preferentially overexpressed in NCCIT
cells, whereas the polycomb target gene sets are underexpressed
(Fig. 2A). The apicidin treatment reverses enrichment patterns of all
gene sets, except Myc targets 2 (Fig. 2A; Supplementary Fig. S4).
Furthermore, the gene sets for Nanog, Oct4, and Sox2 transcription
factors, embryonic stem cell expressions, Nanog, Oct4, and Sox2,
and Nanog targets are markedly affected by apicidin (Fig. 2A). These
findings indicate that the embryonic stem cell-like signature in
NCCIT cells, if not all, could be depleted by HDACI apicidin.
Induction of differentiation and apoptosis of NCCIT cells by
apicidin. To examine the effect of apicidin on induction of
differentiation of NCCIT cells, we have analyzed the expression of
various lineage-specific markers. The quantitative PCR (qPCR)
analysis of mRNA expression reveals that apicidin induces the
expression of the trophectoderm differentiation markers (CDX2
and CG), endoderm markers (GATA4, GATA6, AFP, FOXA2, SOX17,
and CK18), mesoderm markers (BRACHYURY, MSX1, NKX2.5,
NKX3.1, BMP4, HAND1, and MEF2C), and ectoderm markers
(MAP2, PAX6, HES4, and OLIG2; refs. 5, 18), ranging from f5- to
10-fold, compared with control (Fig. 2B), indicating the ability of
apicidin to enhance the differentiation potential of NCCIT cells to
the various lineages. To examine whether apicidin also affects cell
death of NCCIT cells, the effect of apicidin on apoptosis of NCCIT
cells has been analyzed by Annexin V staining using flow
cytometry. Apicidin treatment leads to a significant increase in
the number of Annexin V-positive cells (35.40%) compared with
control (1.60%; Fig. 2C). The similar effect on apoptosis by apicidin
treatment is also observed in NTERA2 and P19 cells (Supplemen-
tary Fig. S5). To further examine the effect of apicidin on
anchorage-independent growth, we performed soft-agar colony-
forming assay, which is considered the most stringent assay for
detecting malignant transformation of cells. Apicidin treatment
dramatically abrogates colony formation of NCCITcells in soft agar
(Fig. 2D). To further prove the role of Nanog in this model, we
performed a rescue experiment by overexpressing Nanog. Over-
expression of Nanog antagonizes the effects of apicidin on cell
cycle arrest and differentiation (Supplementary Fig. S6).
Induction of antiproliferation and differentiation of NCCIT
cells by knockdown of Nanog. To further investigate the
relationship between the biological effects and the down-regulation
of Nanog by apicidin, we have selectively inhibited Nanog
expression using a siRNA targeting Nanog (Fig. 3A). The Nanog-
depleted NCCIT cells show a prolonged doubling time as well as a
significant reduction in bromodeoxyuridine incorporation, com-
pared with the control cells, from 31% to 19% (Fig. 3B and C). To
confirm the role of Nanog in cell growth, we rescued Nanog
expression by replating cells without further transfection of siRNA.
Suppression of cell growth by Nanog knockdown is recovered
similar to that of control cells (Supplementary Fig. S3). In addition,
NCCIT cells transfected with Nanog siRNAs form 62.3% less
colonies than the control transfected cells (Supplementary Fig. S3).
The analysis of lineage-specific markers has revealed that the effect
of knockdown of Nanog on differentiation is similar to that
observed by apicidin treatment (Fig. 3D). However, the Nanog
knockdown has no significant effect on apoptosis (Supplementary
Fig. S3), whereas the proliferative potential is markedly impaired. In
addition, down-regulation of Nanog expression by apicidin
treatment appears not to be attributed to apoptosis, because a
low concentration of apicidin (0.1 Amol/L), which does not induce
apoptosis of NCCIT cells, suppresses the level of Nanog expression.
These results suggest that the effect of apicidin on induction of
differentiation and antitumorigenicity might be mediated by down-
regulation of Nanog but not the effect of apicidin on apoptosis.
Transcriptional inhibition of Nanog by apicidin. Apicidin has
been shown to induce cell cycle arrest at G0-G1in HeLa cells (19).
To elucidate the relationship between apicidin-induced cell cycle
arrest and down-regulation of Nanog, we have analyzed the effect
of apicidin on cell cycle in NCCIT cells. In contrast to G0-G1arrest
in HeLa cells, apicidin induces G2-M cell cycle arrest in NCCITcells
(Supplementary Fig. S5). To examine the possibility that the cell
cycle arrest at G2-M may regulate down-regulation of Nanog, NCCIT
cells were arrested at G2-M by treatment with a pharmacologic
inhibitor nocodazole. As expected, the nocodazole treatment leads
to cell cycle arrest at G2-M, as seen by an increase in the number of
cells at G2-M up to 61% from 30% of control cells, comparable with
that induced by apicidin (Supplementary Fig. S5). However, the G2-
M arrest by nocodazole fails to down-regulate Nanog expression,
indicating that apicidin-induced down-regulation of Nanog expres-
sion might be independent of the apicidin-induced G2-M arrest
(Fig. 4A). In contrast to HDACI, DNA methylation inhibitor 5-aza-
cytidine fails to induce neither cell cycle arrest nor down-regulation
of Nanog (Fig. 4A; Supplementary Fig. S5).
To further characterize the mechanisms by which apicidin
induces down-regulation of Nanog, we examined the recruitment
of RNA polymerase II to the regulatory elements of the Nanog gene.
The chromatin immunoprecipitation analysis using anti-polymer-
ase II (8WG16) antibody shows that apicidin significantly
suppresses the transcriptional initiation, resulting in a decrease
in the transcriptional elongation as seen by a 65% and 68% de-
crease in recruitment of polymerase II into the transcription start
site and coding region of the gene, respectively (Fig. 4B and C).
These results suggest that apicidin might induce down-regulation
of Nanog expression mainly via inhibition of transcription.
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However, down-regulation of Nanog expression by apicidin appears
to be attributed marginally to destabilization of Nanog protein,
because the decrease of in Nanog protein level by apicidin is only
slightly inhibited in the presence of a proteasome inhibitor MG132
(Supplementary Fig. S7).
Induction of repressive epigenetic modifications in the
promoter of Nanog by apicidin. To examine whether down-
regulation of Nanog by apicidin is associated with alterations of
epigenetic modification on the promoter, we have first analyzed
the status of DNA methylation, a repressive epigenetic marker.
Bisulfite mapping reveals that methylation of four CpG sites
located f300 bp upstream of transcription start site (20) is
affected by apicidin treatment. Unmethylated four CpG sites in the
region undergo methylation following apicidin treatment (>30%;
Fig. 4D). We have then performed chromatin immunoprecipitation
analysis to test a possible involvement of histone modifications in
transcriptional suppression of Nanog by apicidin. Apicidin
treatment leads to the significant hypoacetylation of histone H3
and H4 on the region from transcription start site up to f1,000 bp
upstream harboring putative binding sites for transcription factors
such as p53, SP1, Oct4, and Sox2. In addition, active marker K4
trimethylated H3 on nucleosomes associated with the promoter
Figure 2. Apicidin selectively depletes embryonic stem cell-like signature and induces differentiation and apoptosis in NCCITcells. A, cluster of gene sets associated
human embryonic stem cell identity. Among the >2-fold cell significantly changed genes by apicidin (1 Amol/L, 24 h), 1,030 genes were matched with previously
generated embryonic stem cell identity gene sets (datasets S1 and S2). B, effect of apicidin on differentiation in NCCIT cells. Expression levels of lineage-specific
markers were determined by qPCR. C, extent of apoptosis of NCCIT cells in the absence or presence of apicidin (1 Amol/L, 24 h) was determined by flow cytometric
analysis. Representative of three independent experiments. D, soft-agar colony-forming assay with/without apicidin treatment. Colonies were visualized by crystal violet
staining and counted.
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